SPS was supported by the University of Sydney postgraduate research scholarship.

All experimental protocols described were approved by the University of Sydney Animal Ethics Committee (approved protocol number: 2018/1308).
1. Animals
NOTE: Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the Medical Foundation Building Animal Facility at the University of Sydney.
<ol>
	<li>Maintain the mice on a normal 12 h light/dark cycle with environmental enrichment.</li>
	<li>Use male and female C57BL/6 mice (3&#8211;5 weeks old) for all experiments.</li>
</ol>
2. Preparation of Solutions
<ol>
	<li>Prepare 1 L of artificial cerebrospinal fluid (ACSF) composed of 29 mM NaHCO3, 11 mM glucose, 120 mM NaCl, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2.</li>
	<li>Prepare 200 mL of sucrose-ACSF (sACSF) containing 29 mM NaHCO3, 11 mM glucose, 241.5 mM sucrose, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2. Prior to the inclusion of CaCl2 to the ACSF and sACSF, gas the solutions with carbogen (95 % O2 and 5 % CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness).</li>
	<li>Prepare K+-based intracellular solution composed of 70 mM potassium gluconate, 70 mM KCl, 2 mM NaCl, 10 mM HEPES, 4 mM EGTA, 4 mM Mg2-ATP, 0.3 mM Na3-GTP; with a final pH of 7.3 (adjusted using KOH). 
	NOTE: It is recommended to filter intracellular solutions with 0.22 &#181;m filters and store 0.5 mL aliquots of the solution at -20 &#176;C.</li>
</ol>
3. Preparation of the Brainstem
<ol>
	<li>Prior to brainstem extraction, equilibrate the sACSF with carbogen and cool at -80 &#176;C for 25 min so that an ice slurry is formed.</li>
	<li>Anaesthetize the mouse with isoflurane (3&#8211;5 %) saturated in oxygen (3 mL/min). Once the hind paw reflexes are absent, decapitate the mouse with sharp stainless-steel scissors.</li>
	<li>Expose the skull by making a sagittal incision in the skin using a razor blade (#22 rounded).</li>
	<li>Using the pointed end of a pair of standard pattern scissors make a small incision at the lambda and cut along the longitudinal fissure.</li>
	<li>Carefully reflect away the paired parietal bones and the occipital bones using a pair of shallow-bend Pearson rongeurs. 
	NOTE: During this whole procedure the brain is continuously bathed in situ using the previously prepared ice-cold sACSF slurry.</li>
	<li>Isolate the brainstem from the forebrain and its bony encasing using a razor blade (#11 straight) to cut down the parieto-occipital sulcus and at the caudal medulla.</li>
	<li>Mount the isolated brainstem ventral end down on a previously cut trapezoidal polystyrene block. Remove excess fluid around the dissected tissue with a wick of tissue paper to ensure good tissue adhesion to the cutting stage. 
	NOTE: The polystyrene block is cut in a trapezoidal shape, to ensure the rostral end of the midbrain fits and tapers into the spinal cord.</li>
	<li>Use cyanoacrylate glue to fix the polystyrene block with the attached brainstem rostral end down to the cutting stage.</li>
	<li>Using an advance speed of 0.16 mm/s and vibration amplitude of 3.00 mm, prepare 200 &#181;m transverse slices of the MVN. 
	NOTE: Location of the MVN is determined using the Paxinos and Franklin mouse brain atlas (Figures 79&#8211;89)20. The MVN (listed as MVe in atlas) lies immediately ventrolateral to the 4th ventricle and is largest right before the attachment of the cerebellum (between the inferior colliculi and the obex).</li>
	<li>Use a plastic-trimmed pipette to transfer slices onto a filter paper disc sitting in carbogenated ACSF at 25 &#176;C for at least 30 min prior to recording.</li>
</ol>
4. Instruments
<ol>
	<li>Use a standard electrophysiological setup to perform whole-cell patch clamp techniques21.</li>
	<li>Prepare micropipettes using a two-step protocol (heat step 1: 70; heat step 2: 45) on a micropipette puller (see the Table of Materials). Micropipettes should have a final resistance ranging 3&#8211;5 M&#8486; with internal solution when placed in the bath. 
	NOTE: Settings used may vary depending on the temperature within room and can change quite frequently.</li>
</ol>
5. Whole-cell Patch Clamp Electrophysiology
<ol>
	<li>To obtain whole-cell patch clamp recordings from individual neurons in the MVN, a K+-based internal solution is used within the recording pipette.</li>
	<li>Transfer a single tissue slice from the incubation chamber to the recording chamber and secure the slice using a nylon thread on a U-shaped weight. Continuously perfuse the recording chamber with carbogenated-ACSF at 25 &#176;C at a flow rate of 3 mL/min.</li>
	<li>After filling a micropipette with internal solution, locate the MVN using a low power (10x) objective lens. Using a high-power (40x) objective, individual neurons within the MVN can be located. 
	NOTE: Cell quality is essential in ensuring quality recordings and durability of the cell when attempting to achieve the whole-cell configuration. A good cell will demonstrate spherical shape, a reflective cell membrane and an invisible nucleus. A bad cell will have a large visible nucleus (egg-like) and a swollen/shrunken appearance.</li>
	<li>Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip.</li>
	<li>Move the pipette using the micromanipulator towards the chosen neuron and a small dimple should form on the neuronal membrane. Release positive pressure and apply a small amount of negative pressure.</li>
	<li>Once a 1 G&#8486; seal is achieved, apply gentle short and sharp positive pressure to the pipette holder through the suction port to rupture the membrane and create a whole-cell configuration.</li>
	<li>Make whole-cell current clamp recordings using standard techniques21,22.</li>
</ol>
6. Applying Sinusoidal and Stochastic Noise to Individual Medial Vestibular Nucleus Neurons
<ol>
	<li>Apply the stochastic and sinusoidal noise at a range of amplitudes from 3 to 24 pA to determine neuronal threshold and firing rate.</li>
	<li>Determine the sensory threshold by grouping lower and higher stimulus intensities and perform an ANOVA to observe any differences (as shown in Supplementary Figure 1).</li>
	<li>Calculate the average firing rate over the 10 s period where the depolarizing current step was/will be injected for each individual current level (i.e., 7 total episodes; Figure 1).</li>
	<li>Use the average firing rate values to generate a firing rate versus current plot and perform a linear regression analysis to determine the gradient of the line of best fit. The gradient of the line of best fit is indicative of the neuronal gain22.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60044/60044fig1.jpg" /> 
Figure 1: Diagrammatic profiles of control, sinusoidal and stochastic noise protocols. (A) Control (no noise) protocols applied to MVN neurons. (B) Sinusoidal noise protocol with a frequency of 2 Hz. (C) Stochastic noise protocols where majority of the power spectrum is &#8804;2 Hz. Each protocol presented here has an amplitude of &#177;6 pA with a 10 s depolarizing current increasing by 10 pA up to 50 pA. The true stimulus does not have a depolarizing current step and is therefore the first episode of these protocols to determine neuronal gain changes. <a href="https://www.jove.com/files/ftp_upload/60044/60044fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

The effects of galvanic vestibular stimulation (GVS) on the vestibular system has been highlighted in vivo in humans3,13,23, guinea pigs14, rodents18 and non-human primates24. However, none of these studies have assessed the direct impact of electrical noise on the sensitivity of individual neurons in the vestibular system. Here we demonstrate the first in v...

The vestibular (or balance) system controls our sense of balance by integrating auditory, proprioceptive, somatosensory and visual information. Degradation of the vestibular system has been shown to occur as a function of age and can result in balance deficits1,2. However, therapies targeting the functioning of the vestibular system are scarce.
Galvanic Vestibular Stimulation (GVS) has been shown to improve balance measures, autonomic functioning and other sensory modalities within humans3,4,5,6. These improvements are said to be due to the Stochastic Resonance (SR) phenomenon, which is the increase in the detection of weaker signals in non-linear systems via the application of subthreshold noise7,8. These studies have shown improvements in static9,10 and dynamic11,12 balance, and vestibular output tests such as Ocular Counter Roll (OCR)13. However, many of these studies have used different combinations of stimulus parameters such as white noise9, colored noise13, different stimulus frequency ranges and thresholding techniques. Therefore, optimal stimulus parameters remain unknown and this protocol can assist with determining the most effective parameters. Besides stimulus parameters, the type of stimulus is also important in therapeutic and experimental efficacy. The above work in humans was performed using electrical noise stimuli, whilst much of the in vivo animal work has used mechanical14,15 or optogenetic16 noise stimuli. This protocol will use electrical noise to examine the effects on vestibular nuclei.
Previously, application of GVS to stimulate primary vestibular afferents was been performed in vivo in squirrel monkeys17, chinchillas18, chicken embryos15 and guinea pigs14. However, only two of these studies examined the effect GVS has on the gain of primary vestibular afferents14,15. These experiments were performed in vivo meaning that the precise patterns of stimulation imposed on vestibular nuclei cannot be determined. To our knowledge, only one other study has applied stochastic noise to individual enzymatically dissociated neurons in the central nervous system19. However, no experiments have been performed in the central vestibular nuclei to assess appropriate stimulus parameters and thresholding techniques, making this protocol more precise in determining stimulus effects on individual neurons within the vestibular nuclei.
Here, we describe how to apply sinusoidal and stochastic (electrical) noise directly to individual neurons in the medial vestibular nucleus (MVN), determine neuronal threshold and measure changes in gain/sensitivity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl</td>
			<td>Scharlau</td>
			<td>CA01951000</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chem-supply</td>
			<td>PA054-500G</td>
			<td>Used for ACSF, sACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847-100G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187-500MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>Chem-supply</td>
			<td>MA00360500</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Na3-GTP</td>
			<td>Sigma</td>
			<td>G8877-100MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-supply</td>
			<td>SO02270500</td>
			<td>Use for ACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O</td>
			<td>Ajax</td>
			<td>AJA471-500G</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761-1KG</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-supply</td>
			<td>SA030-500G</td>
			<td>Used for sACSF</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>1169567762</td>
			<td>Used for anaesthetising mice</td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Warner instruments</td>
			<td>GC150T-7.5</td>
			<td>1.5mm OD, 1.16mm ID, 7.5cm length</td>
		</tr>
		<tr>
			<td>Data acquisition software</td>
			<td>Axograph</td>
			<td></td>
			<td>Used for electrophysiology and analysis</td>
		</tr>
		<tr>
			<td>Friedmen-Pearson Rongeurs</td>
			<td>World precision instruments</td>
			<td>14089</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td>Used for micropipette</td>
		</tr>
		<tr>
			<td>Multiclamp amplifier</td>
			<td>Axon instruments</td>
			<td>700B</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sper scientific</td>
			<td>860033</td>
			<td>Used for internal solution</td>
		</tr>
		<tr>
			<td>Standard pattern scissors</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Sutter micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225/M</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Used for slicing brain tissue</td>
		</tr>
	</tbody>
</table>

stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed to be due to the stochastic resonance (SR) phenomenon, which is defined as application of a low-level/subthreshold stimulus to a non-linear system to increase detection of weaker signals. However, it is still unknown how SR exhibits its positive effects on human balance. This is one of the first demonstrations of the effects of sinusoidal and stochastic noise on individual neurons. Using whole-cell patch clamp electrophysiology, sinusoidal and stochastic noise can be applied directly to individual neurons in the medial vestibular nucleus (MVN) of C57BL/6 mice. Here we demonstrate how to determine the threshold of MVN neurons in order to ensure the sinusoidal and stochastic stimuli are subthreshold and from this, determine the effects that each type of noise has on MVN neuronal gain. We show that subthreshold sinusoidal and stochastic noise can modulate the sensitivity of individual neurons in the MVN without affecting basal firing rates.

Initial recordings can provide information about the effects that sinusoidal and stochastic noise have on basal firing rates of individual MVN neurons and how the stimuli effect the gain of neurons. Figure 2 shows that neither sinusoidal nor stochastic noise change basal firing rates of MVN neurons when compared to control (no noise) recordings. This information is crucial for determining the threshold of the individual neurons. During the application of galvanic vestibul...

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Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...

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Research

JoVE Journal

Neuroscience

5.0K Views.  University of Sydney. Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Video: Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...